This invention relates to the determination of the glycosylated form of albumin, herein referred to as glycoalbumin, in human blood samples. The determination of the extent of glycosylation of albumin in an individual's blood provides a useful index of glucose level control in diabetics. In particular, the present invention concerns the preparation of monoclonal antibodies which recognize specifically the glycosylated lysine residue at position 525 in human albumin.
Albumin is the major serum protein of blood and has a half life in circulation of 10 days. A non-enzymatic glycosylation reaction results in the covalent coupling of glucose to a small percentage of albumin molecules in all individuals. Since the rate of non-enzymatic glycosylation is dependent upon the circulating level of glucose, diabetics having a higher average level of blood glucose, have an increase in glycosylated albumin. The severity of the diabetic condition is therefore reflected in the percentage of glycosylated albumin.
An analogous reaction occurs between glucose and hemoglobin producing hemoglobin Alc plus other glycosylated hemoglobins. Hemoglobin has a life of 120 days, therefore determination of glycosylated hemoglobin values reflects the average circulating glucose level for that period, whereas a glycoalbumin determination will represent an average circulating glucose level of 10 days. The importance of glycosylated hemoglobin values have been widely accepted as being clinically important for accurate assessment of the diabetic condition. Assays for glycosylated hemoglobin were relatively easy to develop because the hemoglobin molecule is colored and therefore is easy to quantitate using inexpensive spectrophotometers. Albumin is colorless and methods for quantitation of glycoalbumin require that the carbohydrate be derivatized to a colored product or that the protein portion of glycoalbumin be reacted to produce a colored product. For this reason there are no glycoalbumin assays in large scale use in clinical labs at the present time.
A number of proposed glycoalbumin assays are known from the literature. Principal among these are those based on boronate chromatography and thiobarbituric assays. The boronate chromatography method includes the colorimetric determination of bound protein, e.g., bound to Glycogel of Pierce Chemical Co. In this assay, serum is applied to a boronate affinity column wherein all cis-diol containing substances (e.g., glycoalbumin and other glycoproteins) are bound. These substances are then eluted and both bound and eluate fractions quantitated after adding a dye that reacts with proteins producing a colored product. The major disadvantages of this method are that many non-albumin proteins in serum are glycoproteins (e.g., immunoglobulins) and are therefore bound and measured in the boronate chromatography assay; the column procedure has multiple steps for separation and analysis and is not easily automated; and there is also data suggesting that glucose interfers with boronate binding. In the thiobarbituric assay, the ketoamine-protein adduct is converted to 5-hydroxymethylfurfural by hydrolysis with oxalic acid yielding a colored product. The major disadvantages here are that hydrolysis requires 2-4 hours at 100.degree. C. or higher; background color must be corrected; and at the present time, standards or calibrators are not available.
The reaction of glucose with albumin involves (a) the formation of a Schiff's base between C-1 of the glucose with an amino group of albumin and (b) an Amadori rearrangement producing a 1-deoxyfructosyl carbohydrate covalently coupled to the nitrogen of the amino group. The albumin molecule has 60 potential sites (amino groups) for non-enzymatic glycosylation. This is comprised of 59 episilon amino groups of lysine residues and one alpha amino group on the N-terminus of the protein. Of the 60 potential sites only one lysine is known to be glycosylated in the native molecule; however, other lysines may be glycosylated to various extents. The known lysine has been identified as lysine 525 (the 525th amino acid counting from the N-terminus of the protein - Garlick et al, J. Biol. Chem. 258: 6142 (1983)) and the position has been confirmed herein. The reason for the specific glycosylation of this lysine and the rapid rate of glycosylation of albumin is not entirely clear. The specificity for lysine 525 is likely to be (a) the proximity to an adjacent lysine at position 524 thereby lowering the pKa of the .epsilon.-amino group of lysine 525 making it more reactive in the glycosylation reaction, (b) the exposure of the lysine 525 side chain to the aqueous exterior of the albumin molecule, and/or (c) the 3-dimensional structure of the albumin molecule that by an unknown mechanism increases the reactivity of lysine 525 for the glycosylation reaction.
Monoclonal antibodies have been shown to have a precise specificity for binding to a variety of organic compounds, including synthetic peptides. However, despite the availability of this technique and the recognized need for an immunoassay for glycoalbumin, an approach to obtaining antibodies useful for the determination of glycoalbumin has not been reported.
The following definitions will be used herein with respect to amino acid units in peptides.
______________________________________ Definitions Amino Acid Abbreviation ______________________________________ Arginine Arg Aspartic Acid Asp Glutamic Acid Glu Lysine Lys Serine Ser Asparagine Asn Glutamine Gln Glycine Gly Proline Pro Threonine Thr Alanine Ala Histidine His Cysteine Cys Methionine Met Valine Val Isoleucine Ile Leucine Leu Tyrosine Tyr Phenylalanine Phe Tryptophan Trp Alpha-Aminobutyric Acid Aba ______________________________________